ABSTRACT We will develop the first test for detection of viral load (VL) and HIV drug resistance (DR) that is appropriate for low-resource settings (LRS). This new test is based on years of experience developing and testing the Oligonucleotide Ligation Assay (OLA) for HIV DR, as well as our recent work to develop OLA-Simple ? a lab kit that reduces the time and complexity of OLA, but still requires laboratory equipment, laboratory one-way workflow to prevent contamination by amplicons, and hands-on operation of multiple steps. The proposed V- OLA test is a major advance over OLA-Simple that uses innovative technologies coordinated to give a low-cost, streamlined test suite appropriate for HIV patient management. We will develop rapid sample preparation from whole blood, semi-quantitative VL measurement at clinically-relevant thresholds (current WHO cut-off for treatment failure is >1000 copies/mL), and an integrated test cartridge to identify HIV DR mutations (goal to detect 10% mutant for patients with VL >1000 copies/mL). In Aim 1, we will develop chemistry and sample preparation format for HIV RNA isolation compatible with a rapid VL test. In Aim 2, we will develop the VL test using semi-quantitative isothermal amplification, including a simple low-cost heat block and detector. In Aim 3, we will develop core OLA chemistry including isothermal amplification of DR target regions, ligation on single- stranded amplicon, and dry reagents for amplification and OLA; we will test assays on different HIV subtypes. In Aim 4, we will develop the integrated DR detection device, including isothermal amplification and ligation with lateral flow strip detection. In Aim 5, we will combine the VF and DR tests into a coordinated system including refining hardware, scaling up fabrication of kit disposables, and develop a digitally-assisted system ? called Aquarium ? to provide step-by-step guided protocols, sample tracking, results interpretation, and scheduled quality control tests. We will test V-OLA on banked HIV plasma samples (HIV-1 subtypes of A, C, D, and AE). This level of testing is intended to provide preliminary evidence for in-depth studies on performance, usability, and implementation.